This invention is in the fields of pharmacology and neurology. It relates to compounds and methods for protecting the central nervous system against neurotoxic side effects of certain therapeutic drugs and against neurodegenerative disease processes.
Receptors, messenger molecules, agonists, and antagonists
The surfaces of nerve cells in the central nervous system (the CNS, which includes the brain, spinal cord, and retina) contain various types of receptor molecules. In general, a receptor molecule is a polypeptide which straddles a cell membrane. When a messenger molecule interacts with the exposed extracellular portion of the membrane receptor molecule, it triggers a difference in the electrochemical status of the intracellular portion of the receptor, which in turn provokes some response by the cell. The messenger molecule does not bond to the receptor; instead, it usually disengages from the receptor after a brief period and returns to the extracellular fluid. Most receptor molecules are named according to the messenger molecules which bind to them.
An "agonist" is any molecule, including the naturally occurring messenger molecule, which can temporarily bind to and activate a certain type of receptor. An agonist can cause the same effect as the natural messenger molecule, or in some cases it can cause a more intense effect (for example, if it has a tighter affinity for the receptor molecule and remains bound to the receptor for a prolonged period).
By contrast, an "antagonist" is a molecule which can block or reduce the effects exerted by the natural messenger molecule. This can happen in several different ways. A "competitive antagonist" binds to a certain type of receptor without triggering it, thereby preventing the natural messenger molecule from reaching and activating the receptor. A "non-competitive antagonist" functions in other ways. For example, a receptor referred to as the PCP receptor, which is triggered by molecules such as PCP or MK-801, apparently can override the effects of a different type of receptor, the NMDA receptor (both receptors are discussed below). Therefore, PCP and MK-801 are regarded as non-competitive antagonists for the NMDA receptor.
The role a certain molecule plays as an agonist or antagonist must be viewed with regard to a certain type of receptor. For example, while MK-801 is an antagonist for the NMDA receptor, it is an agonist for the PCP receptor. Most agonists and antagonists are xenobiotic drugs, i.e., they do not exist naturally in the body. For more information on neuroanatomy, neurotransmitters, receptors molecules, and agonists and antagonists which interact with CNS receptors, see Adelman 1987 (complete citations are provided below).
The two main classes of excitatory receptor molecules are referred to as "cholinergic" receptors and "glutamate" receptors. Both types of receptors are present in the synaptic junctions that serve as pathways for impulses between CNS nerve cells. Most other types of receptors in the CNS involve inhibitory neurotransmitters.
Excitatory amino acids and neurotoxicity
Accumulating evidence implicates excitatory amino acids (EAA's) such as glutamate and aspartate as causative agents in certain types of CNS damage associated with epsilepsy, hypoglycemia, hypoxia/ischemia (stroke, cardiac arrest, perinatal asphyxia), alcoholism, and trauma of the brain or spinal cord. It is also believed that EAA's may be involved in slowly developing neurodegenerative disorders such as Huntington's, Parkinson's and Alzheimer's diseases. Glutamate and aspartate (the ions or salts of glutamic acid and aspartic acid) are found naturally in high concentrations in the central nervous system (CNS) where they function as excitatory neurotransmitters.
Although these substances are beneficial and of critical importance for the normal functioning of the CNS, under abnormal conditions they can destroy CNS neurons by an "excitotoxic" process. Excitotoxicity refers to the process whereby EAA's that are released from one neuron excessively stimulate (excite) receptor molecules located on the external surface of another neuron. Excitotoxicity also refers to the same excitatory neurotoxic process when triggered by glutamate or EAA analogs of glutamate ingested in foods or administered systemically to various mammalian species. Glutamate and asparate are sometimes called "endogenous" excitotoxins, meaning that they are excitatory neurotoxins contained naturally within the CNS, whereas EAA ingested in foods or administered systemically are referred to as "exogenous" excitotoxins. For an extensive review, see Olney 1989.
EAA receptors, also known as glutamate receptors, are categorized into three subtypes, each named after a glutamate analog which selectively excites them: N-methyl-D-aspartate (NMDA), kainic acid (KA), and quisqualate (QUIS). Glutamate is capable of activating all three receptor subtypes.
Normally, relatively high glutamate concentrations (in the general range of about 10 mM) are maintained inside cells of the CNS, but high concentrations are not allowed in the extracellular fluid where glutamate can exert excitotoxic action at EAA receptors. After glutamate is released by a neuron for neurotransmitter purposes, it normally is transported back inside a cell by means of a transport mechanism which requires energy. Under severe low energy conditions such as hypoglycemia, hypoxia, or ischemia, the transport systems may lack sufficient energy to transfer extracellular glutamate back into the cell, so the glutamate accumulates at abnormal levels and excessively stimulates the EAA receptors. This can lead to continuous neuronal discharge, which in turn causes additional glutamate release and extracellular accumulation of excess glutamate, leading to a cascade of increasing neurotoxic injury, which can result in death or permanent damage to the brain.
Other mechanisms by which EAA's can cause neuronal injury include abnormal sensitivity of EAA receptors to the excitatory action of EAA's, and the presence of abnormal molecules (such as glutamate analogs, certain types of food poisons, etc.) with excitotoxic properties. Such receptor-triggering molecules can accumulate at EAA receptors because they are not recognized by the cellular transport systems as molecules which should be removed from the extracellular fluid.
In these neurotoxic situations, one method of preventing or minimizing excitotoxic injury to the neurons involves administering drugs that selectively block or antagonize the action of the excitotoxic molecules at the EAA receptors.
NMDA antagonists as neuroprotective drugs
The EAA receptor subtype that has been implicated most frequently in neurodegenerative diseases and neurotoxicity is the NMDA receptor. An entire issue of Trends in Neurosciences (Vol. 10, Issue 7, July 1987) was devoted to review articles pertaining to the NMDA receptor, and to NMDA "antagonists" (i.e., molecules which can block or reduce the effects of NMDA at NMDA receptors). Agents which act by binding directly to NMDA receptors, such as D-2-amino-5-phosphonopropanoate (D-AP5) and D-2-amino-7-phosphonoheptanoate (D-AP7), are referred to as competitive NMDA antagonists. Those two compounds are of limited therapeutic utility because they do not readily penetrate the blood-brain barrier. However, it is possible that some recently developed competitive NMDA antagonists, such as the Ciba-Geigy compound CGS 19755 (Boast, 1988) or 3-(2)-carboxypiperazin-4-yl)-propyl-1-phosphonate (CPP) or its unsaturated analog, CPP-ene, may affect the CNS following systemic administration (Herrling et al 1989).
The most powerful and effective NMDA antagonists known at the present time act at another receptor, the phencyclidine (PCP) receptor, which is considered a component of an ion channel complex that involves the NMDA receptor (Kemp et al 1987). These compounds are called non-competitive NMDA antagonists because they do not compete for binding sites at NMDA receptors. When phencyclidine or its analogs activate the PCP receptor, the flow of ions through the NMDA ion channel is blocked or substantially reduced, so that when the NMDA receptor is activated by an EAA, the NMDA receptor response does not result in the flow of ion currents. This blocks the excitation of the neuron.
Four compounds which can activate the PCP receptor, and which therefore serve as non-competitive NMDA antagonists, are phencyclidine, MK-801, ketamine, and tiletamine. Each is discussed in more detail below. All four of these agents can penetrate the blood-brain barrier.
MK-801, a phencyclidine analog manufactured by Merck, Sharp and Dohme (Rahway, N.J.) is believed to be the most powerful PCP agonist of the four compounds listed above (Olney et al 1987). It has generated great interest recently, largely due to its potential for reducing neurotoxic damage involving NMDA receptors. The chemical name for MK-801 is (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate.
Neurological problems that might be aided by NMDA antagonists
Non-competitive NMDA antagonists have been shown in in vivo animal experiments (reviewed in Olney 1989) to protect CNS neurons against damage caused by persistent seizures, hypoglycemia, hypoxia/ischemia, trauma, thiamine deficiency, and methamphetamine poisoning (a form of neurotoxicity related to Parkinson's disease). It is possible, therefore, that such agents might be used therapeutically for neuroprotective purposes in conditions such as the above.
It is also possible that NMDA antagonists might be used to prevent brain damage associated with alcoholism. In chronic alcoholics, neuronal degeneration has been described in several regions of the brain, including periventricular and periaqueductal regions, the thalamus, the hypothalamus, and mammillary bodies. Individuals with this type of brain damage manifest a form of dementia known as Wernicke/Korsakoff syndrome, which includes severe deficits in memory and cognitive functions. It is believed that this syndrome relates to dietary deficiencies, especially thiamine (vitamin B) deficiency. It is known that people who suffer from thiamine deficiency are subject to the same pattern of brain damage and the same dementing Wernicke/Korsakoff syndrome that is seen in alcoholism. Recently, it was demonstrated that in a rat model of thiamine deficiency, which entails disseminated brain damage distributed in a Wernicke/Korsakoff pattern, the brain damage can be markedly attenuated by pretreatment with MK-801 (Langlais et al 1988). That result suggests that this type of brain damage is mediated by an excitotoxic mechanism involving the NMDA receptor ion channel complex, and that patients with acute symptoms suggesting an impending Wernicke/Korsakoff syndrome might benefit from treatment with an NMDA antagonist such as MK-801.
It is also suspected that there may be a link between virally-induced neurodegenerative conditions and NMDA receptor-mediated excitotoxicity (Olney 1989). When a viral infection triggers changes in neural homeostasis, endogenous excitotoxins such as glutamate and aspartate may become involved in cell death or damage. Therefore, NMDA antagonists such as MK-801 may be useful for preventing neuronal degeneration associated with viral infections that involve the CNS, such as Jacob-Creutzfeldt syndrome and encephalitis associated with herpes or measles infection.
NMDA antagonists such as MK-801 might also be useful for protecting against brain damage associated with a newly recognized type of food poisoning. In January 1988, there was an outbreak of food poisoning in Canada affecting 145 identified individuals, some of whom died and were found at autopsy to have widespread brain damage (Quilliam et al 1989). Some of the surviving victims suffered severe brain damage and became permanently demented. All of the victims had ingested mussels from the Newfoundland region. Analysis of the mussels revealed a high concentration of domoate, a powerful convulsant which apparently causes brain damage by inducing persistent seizure activity which releases excessive glutamate, triggering an excitotoxic cascade. It is believed that domoate poisoning may become a recurrent problem in several regions of the world. The inventor of the subject invention has recently demonstrated that certain NMDA antagonists (including MK-801 and phencyclidine) protect against domoate neurotoxicity. Therefore, NMDA antagonists such as MK-801 may serve as antidotes to prevent brain damage, dementia and/or death in domoate poisoning.
In addition to seizure-related brain damage associated with domoate poisoning, brain damage also occurs as a result of persistent seizures in patients with epilepsy. In this situation also, it is believed that excessive activation of NMDA receptors by endogenous glutamate may cause or exacerbate brain damage. It is possible, therefore, that patients who come to emergency rooms in status epilepticus (a state of continuous epileptic seizure activity) might be protected from permanent brain damage by timely treatment with an NMDA antagonist.
However, the potential therapeutic uses of MK-801 and other NMDA antagonists must be viewed with caution, because it has recently been discovered (Olney et al 1989) that such agents can inflict their own type of neurological damage.
The subject invention, as explained below, involves a class of protective agents that can be administered along with NMDA antagonists such as MK-801, to reduce or eliminate the dangers and deleterious side effects of NMDA antagonists. The subject invention thereby enables the safe use of NMDA antagonists such as MK-801 to accomplish the beneficial results set forth above.
Neurotoxic side effects of NMDA antagonists
A potentially serious side effect of MK-801, phencyclidine, and related drugs is that they may induce a neurodegenerative reaction in the posterior cingulate and retrosplenial cerebral cortex, even when administered in relatively low doses (Olney et al 1989). In a series of experiments, MK-801 and phencyclidine were given to adult rats to test for neuroprotection against seizure-related brain damage. Those agents did protect neurons in certain brain regions from seizure-related damage, but they also caused a different type of neurotoxic reaction in other brain regions, the posterior cingulate and retrosplenial cerebral cortices. The neurotoxic reaction, which was observed during microscopic analysis of CNS tissue after the rats were sacrificed, consisted of the formation of vacuoles (membrane-enclosed spaces in the cytoplasm that are not present in normal cells) and the dissolution of mitochondria (energy-producing organelles inside the cells). Although these changes appeared to be reversible if the doses of MK-801 or phencyclidine were sufficiently low, it has recently been discovered that irreversible necrosis of cingulate cortical neurons follows the administration of 5 mg/kg MK-801.
In adult rats, the ED.sub.50 for producing vacuoles in cingulate neurons by MK-801 administration (i.e., the dosage of MK-801 which will produce vacuoles in 50% of the animals treated) is 0.18 mg/kg, administered intraperitoneally (ip; Olney et al 1989). Since the doses of MK-801 used in animal experiments for protecting neurons against ischemic brain damage usually are in the range of 1 to 10 mg/kg, it appears that the use of MK-801 for therapeutic neuroprotection poses a major risk of inducing potentially serious neurotoxic side effects.
The mechanism by which MK-801, phencyclidine and related drugs cause vacuole formation in cingulate/retrosplenial neurons is poorly understood. However, recent evidence that this effect can be reproduced by microinjection of a competitive NMDA antagonist (D-AP5) into the cingulate cortical region (Labruyere et al 1989) suggests that any agent that antagonizes the NMDA receptor ion channel complex by any mechanism may have this toxic property, since the vacuole reaction occurs when NMDA receptor function is suppressed, either by direct antagonist binding of D-AP5 to the NMDA receptor, or by interaction of MK-801 at the level of the NMDA receptor ion channel. An important implication of this finding is that some recently developed competitive NMDA antagonists which may be able to penetrate the brain in sufficient concentration to be used as neuroprotective drugs, such as CGS 19755, CPP, and CPP-ene, may not provide an acceptable alternative to the non-competitive NMDA antagonists, since both groups of compounds might cause the same type of neurotoxic side effect. These findings and implications suggest that both groups of compounds would be more acceptable for therapeutic purposes if a method were found that prevents their neurotoxic side effects without interfering with their neuroprotective actions.
History of the uses and abuses of NMDA antagonists
Phencyclidine (PCP) was originally introduced into clinical medicine some 30 years ago as an anesthetic (Goodman and Gilman 1975). Shortly thereafter, it was withdrawn from the market because it was found to have hallucinogenic properties which invited illicit use by drug abusers. Since then, PCP (also known as angel dust) has become increasingly popular as a "recreational" drug and currently is a major cause of drug-induced psychotic reactions (which occasionally lead to extremely violent crimes) among drug abusers. The pathomorphological effect of PCP (vacuole formation and mitochondrial dissolution in certain types of neurons) might be related to the mechanism by which PCP causes toxic psychoses. Thus, if a drug could be found that prevents the pathomorphological effects of PCP, it might also prevent or ameliorate the psychotomimetic effects of PCP. Such a drug might be used in emergency rooms, or perhaps by the police, as an antidote to reduce both the neurological damage and the psychotic effects of PCP in drug abusers.
Ketamine, a drug manufactured by Parke Davis and marketed under the trade name Ketalar, is currently used both in human and in veterinary medicine as an anesthetic. Ketamine is known to activate PCP receptors and, like PCP and MK-801, is recognized as a non-competitive antagonist of the NMDA receptor-ion channel complex (Kemp et al 1987). Ketamine was among the drugs recently shown to produce pathomorphological effects on cingulate/retrosplenial neurons following intraperitoneal (ip) administration to rats (Olney et al 1989). Ketamine is known to induce an acute transient psychosis (called an "emergence" reaction) in about 13% of human patients anesthetized with this agent (Physicians Desk Reference, 1986). It has been proposed that the psychotic effects of ketamine, like those of PCP, may be psychological manifestations of the same toxic process that causes pathomorphological changes in cingulate/retrosplenial neurons, in which case a drug that could prevent the pathomorphological changes might also prevent or reduce the psychotic manifestations. Even without preventing the psychotic manifestations, eliminating the risk of pathomorphological changes would be a significant benefit.
Tiletamine is a drug manufactured by A. H. Robins. It is currently used in veterinary medicine, and is widely used for anesthesia on house pets. Tiletamine, like PCP, MK-801 and ketamine, is known to activate PCP receptors and is recognized as a non-competitive antagonist of the NMDA receptor-ion channel complex. Tiletamine was among the drugs recently shown to produce pathomorphological effects on cingulate/retrosplenial cortical neurons following ip administration to rats (Olney et al 1989).
MK-801, a drug manufactured by Merck, Sharp and Dohme and referred to as dizocilpine, was initially proposed as an anticonvulsant, but after brief human clinical trials, it was withdrawn from further testing several years ago with no published explanation. At about the same time, it was discovered that MK-801 is a potent activator of PCP receptors (with higher affinity for the PCP binding site than PCP itself; MK-801 is more specific for PCP receptors than any other known compound), and that PCP receptors are an integral component of the NMDA receptor ion channel complex (Kemp et al 1987). It was also found that both MK-801 and PCP block the excitatory effects of NMDA on neurons in the in vivo rat spinal cord (Lodge et al 1987). In neurotoxicology studies, using an ex vivo chick embryo retina assay, it was shown that MK-801 is approximately 5-10 times more powerful than PCP in preventing the neurotoxic effects of NMDA on retinal neurons (Olney et al 1987). Previously, PCP had been recognized as the most powerful known antagonist of NMDA neurotoxicity (Olney et al 1986).
Because of its great potency and the ease with which it penetrates blood brain barriers, MK-801 has become the drug used most widely in animal experiments aimed at testing the neuroprotective properties of NMDA antagonists. Since it has now been shown to protect CNS neurons against various degenerative processes that are thought to involve excessive activation of NMDA receptors (e.g., hypoxia/ischemia, prolonged seizures, hypoglycemia, thiamine deficiency, head or spinal cord trauma) there is considerable interest in using MK-801 for neuroprotective purposes in clinical neurology. Clearly, it would be desirable to have a means of preventing the toxic action of MK-801 on cingulate/retrosplenial cortical neurons, thereby making this drug available for human therapy with reduced risk of neurotoxic side effects.
Cholinergic receptors
Cholinergic receptors are activated by acetylcholine, a relatively small molecule released by certain types of brain cells. Cholinergic receptors are divided into two main classes: muscarinic and nicotinic.
Little is known about nicotinic receptors in the CNS. They exist in the peripheral nervous system, at neuromuscular junctions, and they are presumed to exist inside the brain, but very limited progress has been made in developing agonist or antagonist molecules that can penetrate blood-brain barriers and be used to pharmacologically characterize nicotinic receptors that may exist in the brain.
Muscarinic receptors are subdivided into M1 and M2 receptors, based on the discovery that pirenzepine binds with much greater affinity to one subpopulation (M1) found primarily in the forebrain, than to a separate subpopulation (M2) that exists primarily in the hindbrain and in the peripheral nervous system. Most anti-cholinergic molecules, although more powerful than pirenzepine in binding to cholinergic receptors, do not show as high a degree of specificity for one receptor subpopulation. Thus, the anti-cholinergic agents of primary interest herein have substantial affinity for both M1 and M2 receptors (Burke 1986; Freedman et al 1988). It is not known whether or to what extent these anti-cholinergics also interact with nicotinic receptors inside the brain, since reliable methods for identifying and characterizing nicotinic receptors in the brain have not been available.
Pilocarpine, a cholinergic agonist used in epilepsy research, has been shown to cause seizures and seizure-related brain damage (Turski et al 1983; Clifford et al 1987). Although the inventor has used MK-801 successfully to prevent brain damage associated with seizures induced by various methods, a set of experiments described in Example 5 indicates that MK-801 has a potentiating effect when administered along with pilocarpine. The term "potentiate" refers to the fact that the MK-801 lowered the seizure threshold and made test animals susceptible to seizures at a pilocarpine dosage that would not have caused seizures in the absence of the MK-801. This finding raises questions about whether NMDA antagonists would tend to induce seizures in humans who suffer from epilepsy.
The inventor also discovered that pilocarpine and MK-801, when administered together, increase the formation of vacuoles in cingulate/retrosplenial neurons. If an adult rat is treated with pilocarpine (75 mg/kg ip), the ED.sub.50 of MK-801 for producing cingulate/retrosplenial vacuoles is reduced from 0.18 mg/kg to 0.05 mg/kg.
Judging from these results, MK-801 apparently can exert one type of beneficial anti-convulsant effect, by blocking one of the major excitatory transmitter systems, the NMDA receptor system. However, MK-801 appears to potentiate another type of seizure activity mediated by the cholinergic receptor system. These results imply that some kind of mechanism exists by which the cholinergic and NMDA receptor systems are linked, such that drugs affecting either system can influence neurological disorders such as seizure activity and formation of vacuoles in cingulate/retrosplenial neurons.
Anti-cholinergic agents
A group of agents classified as anti-cholinergics (i.e., they block the activation of cholinergic receptors) have been used in clinical neurology as anti-parkinsonian drugs (Goodman and Gilman 1975). These agents were recently found by the inventor to protect rats against the convulsant and brain damaging action of pilocarpine and another cholinergic neurotoxin, soman (Price et al 1989). The drugs that conferred this neuroprotective action are procyclidine, biperiden and trihexyphenidyl, which are structurally related compounds of the aryl-cyclo-alkanolamine class. These agents, especially biperiden and trihexyphenidyl, are considered cholinergic antagonists that act quite powerfully at the M1 muscarinic receptor (Freedman et al 1988), which suggests a possible explanation for their efficacy in blocking the neurotoxic actions of pilocarpine, which is primarily an M1 cholinergic agonist. These aryl-cycloalkanolamines have also been shown to have limited effectiveness as NMDA receptor antagonists (Olney et al 1987), but they are considered much more powerful as M1 cholinergic antagonists than as NMDA antagonists.
Procyclidine, which has some degree of affinity for NMDA receptors in addition to being an anti-cholinergic agent, can be administered to adult rats at a high dose (75 mg/kg) without producing neurotoxic side effects such as cingulate/retrosplenial vacuole formation induced by other NMDA antagonists such as MK-801 or D-AP5. Procyclidine is described in U.S. Pat. No. 2,891,890 (Adamson 1959), and is marketed under the trade name "Kemadrin" by Burroughs-Wellcome.
Biperiden has been studied for its mood altering effects (Fleischhacker et al 1987) and for its interaction with muscarinic receptors (Syvalahti et al 1987). The hydrochloride salt of biperiden has been studied for its interaction with nicotine and oxotremorine in rat diaphragm (Das et al 1977). Biperiden is marketed under the trade name "Akineton" by Knoll. Triperiden is marketed in Europe under the trade name "Norakin" by VEB Fahlberg-List (Magdeburg, West Germany).
Trihexyphenidyl has been studied for its effects in schizophrenic patients (Hitri et al 1987) and for its effects on memory in elderly patients (McEvoy et al 1987). It is marketed under the trade name "Artane" by Lederle, and is used to reduce Parkinson symptoms in schizophrenics who are being treated with phenothiazine compounds.
Various other aryl-cycloalkyl-alkanolamine compounds have also been studied for varying purposes (e.g., U.S. Pat. Nos. 4,031,245 and 3,553,225, West German Offen. No. 1,951,614, and Mann et al 1976). However, none of the research with this class of compounds involves their use for reducing the neurotoxic effects of PCP, MK-801, or other NMDA antagonists.
A number of other compounds are known to function as anti-cholinergic agents. Benztropine, sold under the trade name "Cogentin" by Merck, Sharp and Dohme, is used to reduce Parkinson symptoms in schizophrenics being treated with phenothiazine compounds (Vernier 1981). Benactyzine is used in conjunction with meprobamate in a formulation called "Deprol," sold by Wallace Laboratories (Physicians Desk Reference 1989, p. 2200). Scopolamine and atropine, both of which have been used widely in medicine for anesthesia-related purposes, have also been used as anti-Parkinsonian drugs, but they tend to cause side effects when administered at anti-Parkinson dosages, due to their interactions with cholinergic receptors in the peripheral nervous system. In summary, accumulating evidence suggests that NMDA antagonists might be highly useful therapeutic agents in various neurological disorders. However, prior to this invention, there was no known agent or method for preventing certain deleterious side effects of those NMDA antagonists.
One object of this invention is to provide a pharmacological agent and method which can be used in human medicine to reduce the neurotoxicity of NMDA antagonists.
Another object of this invention is to provide an agent and a method for reducing the neurotoxicity of agents such as ketamine and tiletamine, which are used as veterinary anesthetics on animals including house pets.
Another object of this invention is to provide a mixture of an NMDA antagonist combined with a protective agent which reduces the neurotoxic effects of the NMDA antagonist. Such mixtures can be used to prevent or minimize deleterious CNS effects associated with various neurological disease processes.